Cytoplasmic, nuclear and mitochondrial protein extraction

HL-60 cells (2×10⁶ cells/ml) in the logarithmic growth phase were harvested and washed with ice-cold phosphate-buffered saline (PBS). Nuclear and cytoplasmic proteins were isolated from cells using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. Briefly, cells were transferred to a 1.5 ml microcentrifuge tube and pelleted by centrifugation at 500 × g for 5 min, 4°C before a pipette was used to remove the supernatant. Ice-cold Cytoplasmic Extraction Reagent (CER) I was subsequently added to the cell pellet, and the tube was vortexed vigorously for 10 sec before incubating on the ice for 20 min. Ice-cold CER II, was added to the solution, and the tube was vortexed for 5 sec and then centrifuged for 10 min, 4°C at maximum speed in a microcentrifuge (160,000 × g). The resulting supernatant was the cytoplasmic extract, and the insoluble fraction, containing the cell nuclei, was dissolved in ice-cold Nuclear Extraction Reagent. The sample was subsequently vortexed for 15 sec and then incubated on ice for 10 min. This step was repeated 4 times over the course of 40 min and was followed by centrifugation at maximum speed (160,000 × g) in a microcentrifuge for 30 min at 4°C, to obtain the nuclear protein extract.

Mitochondrial proteins were isolated using a Cell Mitochondria Isolation kit (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's instructions. Briefly, cells (2×10⁶ cells/ml) in the logarithmic growth phase were harvested and washed twice with ice-cold PBS. Cells were incubated with cell lysis buffer (1 ml cell lysis buffer/2×10⁷ cells) for 10 min at 4°C, before they were homogenized with a glass homogenizer. The cell lysate was centrifuged at 600 × g for 10 min at 4°C to remove any remaining whole cells, and the supernatant was further centrifuged at 11,000 × g for 10 min at 4°C. The supernatant was removed and the remaining mitochondrial pellet was resuspended in mitochondrial lysis buffer at 4°C, and centrifuged at 24,000 × g for 15 min at 4°C to remove the cell nuclei. Mitochondrial extracts were stored at −80°C until required. The concentration of cytoplasmic, nuclear and mitochondrial protein extracts was determined using a bicinchoninic acid assay kit (Pierce; Thermo Fisher Scientific, Inc.).